GRAPHICAL ABSTRACT

INTRODUCTION

Nonalcoholic fatty liver disease (NAFLD) is the most widespread liver disease in the world [1]. It has a prevalence rate of 33% in the general population and 70% to 75% in the obese and diabetic patient population in Western countries [2,3]. Particularly, obesity is a major public health issue because it provokes metabolic syndromes including hypertension, type 2 diabetes mellitus, NAFLD, and insulin resistance [4]. Insulin resistance plays a pivotal role, by increasing the influx of free fatty acids (FFAs) to the liver, in the development of hepatic steatosis [5,6]. While early-stage steatosis is typically considered benign, later-stage nonalcoholic steatohepatitis (NASH) is destructive, resulting in the death of hepatocytes and progression to fibrosis and cirrhosis [7].

Hepatic steatosis is caused by increased influx of FFAs, such as palmitic acid (PA), in liver tissues. Sterol regulatory element-binding protein-1c (SREBP-1c) is an essential transcription factor that regulates lipid synthesis in insulin-sensitive tissues, such as the liver, muscle, and adipose tissue [8]. Aberrant activation of SREBP-1c leads to excessive lipid accumulation and insulin receptor substrate 1 (IRS-1) suppression, resulting in increased muscular insulin resistance [9]. The activity of SREBP-1c is known to be elevated in insulin and nutrient-rich conditions, whereas its activity is suppressed by AMP-activated protein kinase (AMPK) [10,11]. Recent studies showed that serine/threonine kinase Rho-kinase 1 (ROCK-1), a downstream effector of the small G protein RhoA, was activated by PA. Additionally, ROCK1 knockdown recovered the downregulation of AMPK phosphorylation in PA-stimulated L6 myotubes, indicating that ROCK is related to the effect of insulindependent AMPK action [12]. In summary, PA-mediated ROCK activation inhibits AMPK activity, resulting in increased expression of lipogenesis-related genes, such as Srebp-1c and fatty acid synthase (Fas).

Increased levels of circulating serum FFA and accumulation of hepatic lipid in non-adipose tissues can lead to hepatocellular dysfunction or cell death, partly because of the diversion of unoxidized FFAs to nonoxidative pathways, resulting in lipoapoptosis [13]. Excess amounts of nonesterified free fatty acids (NEFFAs) that have not been converted to triglyceride (TG) in hepatocytes, promote hepatic lipotoxicity, and its pathologic feature is observed in NASH [14]. The mechanisms involved in FFA-induced lipotoxicity are not clearly known. Nevertheless, recent studies demonstrate that hepatic lipoapoptosis mainly arises from FFA-induced lipotoxic stress of intracellular organelles, such as the endoplasmic reticulum (ER) and mitochondria [15]. Therefore, saturated FFAs, especially PA, have been implicated in ER stress-mediated apoptosis of hepatocytes [16,17]. Protein kinase R-like endoplasmic reticulum kinase (PERK), one of the major transducers of ER stress, phosphorylates α subunit of eukaryotic initiation factor 2 (eIF2α) on Ser 51, and phosphorylated eIF2α suppresses the initiation of general translation to decrease the protein load in the ER [18]. Conversely, activated eIF2α elevates in the translation of the activating transcription factor 4 (ATF4), and activated ATF4 increases the expression of C/EBP-homologous protein (CHOP), and the CHOP in turn inhibits anti-apoptotic B-cell lymphoma 2 (Bcl2) expression [19,20]. Accordingly, the ER stress-mediated PERK/eIF2α/ATF4/CHOP axis signaling pathway is heavily involved in PA-induced lipoapoptosis.

Liver fibrosis is caused by excessive accumulation of extracellular matrix proteins such as collagen, fibrin, and fibronectin, as seen in most chronic liver diseases. Progression of liver fibrosis results in cirrhosis, hepatic failure, and portal hypertension. Halofuginone (HF) is an analog of alkaloid febrifugine originally isolated from the plant Dichroa febrifuga. In recent years, HF has attracted much attention because of its wide range of beneficial biologic activities, such as in malaria, cancer, and particularly in fibrosis-related diseases. Recently, HF has been reported to be involved in two functional mechanisms, namely inhibition of the transforming growth factor β (TGFβ)/smad3 signaling pathway [21] and inhibition of prolyl-tRNA synthetase (PRS) activity [22]. Treatment with HF decreases TGFβ1-induced collagen synthesis in fibroblasts [23] without affecting TGFβ receptor expression or TGFβ product levels [24], indicating that HF targets TGFβ receptor 1 (TGFβR1)-mediated SMAD3 phosphorylation [25]. Furthermore, PRS has been found to block collagen synthesis and production [26]. HF inhibits the mRNA expression levels of TGFβ1-increased collagen I a1 (Col1a1) and collagen I a2 (Col1a2). However, their levels are recovered by the addition of exogenous proline [26], indicating that HF competitively binds to the proline-binding pocket of the PRS catalytic site [27], which involves the loading of proline to the tRNA during the translational process of proline-rich profibrotic genes, such as Col1a1 and fibronectin (Fn1). Some previous studies reported that proline contents are higher in fibrotic tissues than in non-fibrotic tissues [28]. Therefore, proline accumulation in fibrotic tissues could decrease via the anti-fibrotic effect of HF. Despite its positive effects, several adverse effects of high doses of HF have been reported, such as cytotoxicity, organ failure, and gastrointestinal toxicity [29]. DWN12088 (Daewoong Pharmaceutical, Seoul, Korea), a first-in-class PRS inhibitor, which is a novel therapeutic agent for idiopathic pulmonary fibrosis (IPF), showed anti-fibrotic properties, safety, and tolerability in a phase 2 trial [30]. However, the mechanisms underlying the hepatoprotective effect of DWN12088 have not yet been studied in vitro and in vivo. In this study, we aimed to determine whether DWN12088 alleviates hepatic injury in NASH.

METHODS

Chemicals, plasmids, and materials

DWN12088 was obtained from Daewoong Pharmaceutical Co., Ltd. HF was purchased from (Calbiochem, San Diego, CA, USA). Palmitate was purchased from Sigma-Aldrich (St. Louis, MO, USA), and it was conjugated to fatty acid-free bovine serum albumin (BSA) (Sigma-Aldrich) as previously described [31]. MG132, thiazolyl blue tetrazolium blue (MTT), and Oil red O were purchased from Sigma-Aldrich. Boron-dipyrromethene (BODIPY) was obtained from Invitrogen (Carlsbad, CA, USA).

Mice experiments

Male 8-week-old C57BL/6J mice were purchased from Dae Han Bio Link Co. (Eumseong, Korea) and allowed to adapt for 1 week. All mice were housed in a cage with a 12-hour lightdark cycle in a pathogen free animal facility. At 9 weeks old, the mice were fed a chow or methionine-choline deficient (MCD)-diet, administrated with DWN12088 (10 mg/kg, every 2 days apart, n=10 mice per group) or saline (equal volume, every 2 days apart, n=10 mice per group) by oral gavage for 6 weeks. The body weights of the mice were recorded weekly. After 15 weeks, the liver tissues and serum were collected and stored at –80°C for later assessment. All animal experiments were approved by the Institutional Animal Care and Use Committee of Yonsei University at the Wonju Campus (YWC-200401-1). All experimental procedures were performed in accordance with the guidelines of institutional animal care.

Cell culture and treatments

Mouse normal hepatocyte cell line alpha mouse liver 12 (AML12) were maintained in 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 (Corning Inc., Corning, NY, USA), containing 10% fetal bovine serum, 100 units/mL of penicillin, 100 µg/mL of streptomycin, 40 ng/mL of dexamethasone, and 1× insulin-transferrin-selenium cocktail (Gibco, Grand Island, NY, USA). Human hepatic stellate cells (HSCs) LX-2 were cultured in DMEM supplemented with 10% fetal bovine serum and 100 units/mL of penicillin. Mouse primary peritoneal macrophages were collected from the peritoneal cavity of 6- to 8-week-old mice as previously described [32]. Peritoneal macrophages and RAW264.7 cells of the murine macrophage cell line were cultured in DMEM, containing 5% fetal bovine serum supplied with 100 units/mL of penicillin, 100 µg/mL of streptomycin, and 25 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The cells were incubated at 37°C in a 5% CO₂ humidified incubator. Experiments were performed when the cells had reached 60% to 80% confluence.

Cell viability

The details on measurement of cell viability are described in the ‘Supplementary Methods’ section.

RNA isolation and real-time quantitative reverse transcription polymerase chain reaction

The details on RNA isolation, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), and primer information are provided in the ‘Supplementary Methods and Supplementary Table 1’ section.

Immunoblotting

Cell and tissue lysates as well as cytosolic and nuclear extracts were prepared as previously described [33]. Equal amounts of total protein were separated by 8% to 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes using a transfer device. The blots were blocked with 3% BSA or 5% skim milk in Tris-buffered saline (T&I, Seoul, Korea) containing 0.1% Tween 20 (Sigma-Aldrich) for 1 hour and immunoblotted with the indicated antibodies. The antibodies information is listed in the ‘Supplementary Methods’ section.

Luciferase reporter assay

The details on experimental procedures are explained in the ‘Supplementary Methods’ section.

Immunocytochemical and histological examination

The details on experimental procedures and material information are provided in the ‘Supplementary Methods’ section.

Hematoxylin and eosin, Masson’s trichrome, Oil red O, and BODIPY staining

The details on experimental procedures are provided in the ‘Supplementary Methods’ section.

NAS scoring

Total NAFLD activity score (NAS) was calculated as the sum of steatosis, lobular inflammation, and hepatocyte ballooning scores in the hematoxylin and eosin (H&E)-stained liver section slides. The NAS score range (0–8) was calculated by a sum of steatosis (0–3), lobular inflammation (0–3), and hepatocyte ballooning (0–2). The scores were blindly analyzed in eight fields per slide section.

TUNEL assay

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed on liver tissue sections. The section slides were analyzed using an in situ Cell Death Detection Kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. All the slides were mounted with mounting medium and observed using a Zeiss LSM710confocal microscope (Carl Zeiss, Berlin, Germany) at ×400 magnification. The percentage of TUNEL-positive cells was determined for at least four fields per section in a randomized manner using the ImageJ software (National Institutes of Health, Bethesda, DM, USA).

Biochemical analysis

Levels of TG, cholesterol, FFAs, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured using a colorimetric assay kit (Asan Pharm., Seoul, Korea), according to the manufacturer’s instructions. Hydroxyproline content in the liver tissue was analyzed using a hydroxyproline assay kit (Cell Biolabs, San Diego, CA, USA), according to the manufacturer’s instructions.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA). The number of replicates is listed in the figures and the representative results were obtained from at least three independent experiments. All values are presented as the mean±standard deviation. The statistical significance was evaluated using an unpaired two-tailed t-test between two groups or one-way or two-way analysis of variance (ANOVA) followed by the Holm-Sidak multiple comparison test, depending on the experimental groups analyzed. A P value of <0.05 was considered statistically significant.

RESULTS

High-dose halofuginone reduces cell viability, but not DWN12088

HF, an analog of alkaloid febrifugine, is an anti-fibrotic agent that suppresses the transcriptional and translational processes of proline-rich profibrotic genes, such as COL1A1, COL1A2, and FN1 [34,35]. However, recently, HF has been reported to cause several adverse effects, such as poor oral bioavailability and gastrointestinal toxicity [29]. Similarly, DWN12088, a therapeutic agent for IPF, is a selective PRS inhibitor that inhibits the synthesis and production of collagen. To compare the cytotoxic effects of HF and DWN12088, mouse normal hepatocytes AML12 and human HSCs LX-2 were treated with indicated dose conditions of HF and DWN12088 for 24 hours. The cell viability was decreased by HF in a dose-dependent manner, but not by DWN12088 (Fig. 1A and B). As expected, active-caspase 3 expression was markedly increased by 0.3 μM HF, but not by the high-dose of DWN12088 (Fig. 1C and D). Of note, high-dose DWN12088 was not as cytotoxic as high-dose HF, indicating the suitability of DWN12088 as a new anti-fibrotic drug.

DWN12088 decreases profibrotic gene expression and hepatic fibrosis in TGFβ-treated HSCs and liver of MCD-diet mice

To assess whether DWN12088 could suppress in vitro liver fibrosis, we first examined the expression levels of profibrotic marker genes, such as collagen I, fibronectin, and α-smooth muscle actin (α-SMA), in LX-2 cells pretreated with or without DWN12088 after TGFβ stimulation. DWN12088 treatment crucially decreased TGFβ-induced profibrotic marker gene expressions, which were confirmed by immunoblotting and immunofluorescence staining (Fig. 2A and B). Similarly, DWN12088 markedly inhibited TGFβ-dependent COL1A1, COL1A2, FN1, and actin alpha 2 (ACTA2) mRNA expressions (Fig. 2C). TGFβ evokes fibrosis by upregulating the TGFβR1/Smad2/3 and TGFβR1/glutamyl-prolyl-tRNA synthetase (EPRS)/signal transducer and activator of transcription 6 (STAT6) signaling cascades [36,37]. Phosphorylated smad2/3 and STAT6 causes transcriptional activations of the promoters of fibrosis-related genes, such as COL1A1, FN1, and ACTA2. We identified the anti-fibrotic effects of DWN12088 involved in the signal modulators. TGFβ treatment increased the phosphorylation of smad2/3 and STAT6 and expression of EPRS, whereas TGFβ and DWN12088 co-treatment downregulated TGFβ signal modulators, such as smad2/3, STAT6, and EPRS in LX-2 cells (Supplementary Fig. 1). Additionally, the similar effects of DWN12088 were also observed in AML12 hepatocytes (Supplementary Fig. 2). Next, we assessed whether DWN12088 treatment decreases MCD-diet-induced liver fibrosis. Hepatic hydroxyproline contents and Masson’s trichrome staining showed that compared to that with saline treatment, DWN12088 significantly attenuated MCD-diet-induced collagen fiber formation in liver tissues (Fig. 2D and E). As expected, DWN12088 also drastically inhibited MCD-diet-induced protein and mRNA levels of Col1a1, Col1a2, Fn1, and Acta2 (Fig. 2F and G). These results suggest that DWN12088 effectively ameliorates hepatic fibrosis by inhibiting the TGFβ signal modulators-mediated upregulations of profibrotic marker gene expressions.

DWN12088 attenuates lipid accumulation by inhibiting ROCK/AMPK/SREBP-1c signaling cascades in palmitic acid-stimulated hepatocytes and liver of MCD-diet mice

To explore whether DWN12088 reduces lipid accumulation, AML12 cells were treated with PA alone or with DWN12088 (5 μM, 10 μM) for 24 hours. Oil red O staining and TG contents showed that DWN12088 effectively inhibited PA-induced lipid accumulation in a dose-dependent manner (Fig. 3A and B). SREBP-1c plays a pivotal role in the transcriptional activation of fatty acid synthesis-related genes. PA-activated ROCK blocks AMPK activity, resulting in upregulated SREBP-1c and FAS expressions [12]. Therefore, we examined the inhibitory effects of DWN12088 on PA-activated ROCK/AMPK/SREBP-1c signaling pathways. DWN12088 treatment significantly suppressed the activity of PA-induced signal modulators in a dose-dependent manner. Moreover, PA increased the levels of lipogenesis marker proteins FAS and adipophilin (ADRP) and diminished the levels of lipolysis marker proteins peroxisome proliferatoractivated receptor α (PPARα) and peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α), but these effects were dose-dependently reversed by the DWN12088 (Fig. 3C). Similarly, DWN12088 reversed mRNA expressions of PAregulated lipid synthesis-related genes, such as Srebp-1c, Fas, Adrp, Ppara, and Pgc1a (Supplementary Fig. 3A). Next, we investigated whether DWN12088 treatment attenuates MCD-diet-induced hepatic steatosis. Picture data of liver morphology showed that DWN12088 markedly diminished MCD-diet-induced hepatic steatosis compared to that with saline treatment (Fig. 3D). Compared with that of the chow group, the MCD-diet group lost body and liver weights. However, the body and liver weights did not differ between the saline- and DWN12088-administrated groups (Fig. 3E). There was however no significant difference between the hepatic TG levels of the mice within the same treatment group (Supplementary Table 2). Based on this, five mice were selected as representative samples for each group and Oil red O (Supplementary Table 3) and BODIPY staining was conducted. The Oil red O and BODIPY staining revealed that DWN12088 substantially suppressed MCD-diet-induced lipid accumulation in liver tissues compared to that by saline (Fig. 3F and G). Moreover, similar effects were also confirmed by biochemical analysis of hepatic TG, serum TG, total cholesterol, and serum FFA levels (Fig. 3H). We then confirmed whether DWN12088 attenuated MCD-diet-induced lipid synthesis-related proteins, such as FAS, ADRP, and acetyl-CoA carboxylase α (ACCα) by inhibiting the ROCK/AMPK/SREBP-1c signaling pathway. As expected, compared to that with saline treatment, DWN12088 treatment significantly decreased MCD-diet-induced lipid accumulation-related signal modulators and marker proteins in the liver tissues (Fig. 3I and J). Furthermore, MCD-diet-increased mRNA levels of lipogenesis-related genes, such as Srebp-1c, Fas, and Adrp, and MCD-diet-inhibited mRNA levels of lipolysis-related genes, such as Ppara, Pgc1a, and carnitine palmitoyltransferase 1A (CPT1a) were recovered by DWN12088 (Supplementary Fig. 3B). Taken together, these data suggest that DWN12088 has a protective effect against PA-stimulated lipid accumulation and MCD-diet-induced hepatic steatosis.

DWN12088 mitigates inflammatory responses by inhibiting NF-κB activation in palmitic acid-stimulated macrophages and liver of MCD-diet mice

AMPK signaling can inhibit NF-κB-induced inflammatory responses [38]. PA may be suppressed in the phosphorylation of AMPKα at Thr172 via the increase of ROCK expression, leading to NF-κB activation. We assessed whether DWN12088 suppresses PA-induced NF-κB activation by inhibiting ROCK signaling cascades in murine macrophages RAW264.7 cells. As expected, DWN12088 treatments markedly restored PA-inhibited AMPK phosphorylation via inhibition of ROCK expression in a dose-dependent manner (Fig. 4A). Moreover, DWN12088 significantly decreased PA-mediated NF-κB activation by increasing NF-κB p65 nuclear translocation via phosphorylation and degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) (Fig. 4B). Additionally, phosphorylation of NF-κB p65 at Ser 536 and Ser 276 in the cytosol and nuclei, NF-κB-driven luciferase activity, and NF-κB p65 nuclear translocation revealed that DWN12088 significantly inhibited PA-induced NF-κB activation (Fig. 4C-E). Furthermore, PA-induced mRNA expressions of proinflammatory genes, such as tumor necrosis factor-α (Tnfa), interleukin 1β (Il1b), and Il6 and PA-inhibited mRNA expressions of anti-inflammatory genes Il10 and arginase 1 (Arg1) were reversed by DWN12088 in the RAW264.7 cells (Fig. 4F). Similar effects of DWN12088 on mRNA expressions of inflammatory genes were also observed in peritoneal macrophages (Supplementary Fig. 4). To evaluate whether DWN12088 inhibited MCD-diet-induced steatohepatitis, we first analyzed the infiltration of M1 proinflammatory macrophages in the liver tissues. Immunofluorescence staining and qRT-PCR analysis have shown that DWN12088 markedly decreased MCD-diet-induced infiltration of M1 proinflammatory macrophages and mRNA levels of M1 macrophage marker F4/80, also known as EGF-like module-containing mucin-like hormone receptor-like 1 (Emr1) (Fig. 4G and H). In addition, DWN12088 treatment attenuated MCD-diet-induced IκBα degradation and p65 NF-κB phosphorylation, resulting in increased NF-κB activation (Fig. 4I and J). As expected, mRNA levels of NF-κBdependent proinflammatory genes, such as Tnfa, Il1b, Il6, and CC motif chemokine ligand 2 (Ccl2) were significantly increased, and expressions of anti-inflammatory genes, such as Il10 and Arg1, were inhibited in the liver tissues of saline-treated MCDdiet group, whereas the expressions were reversed in the DWN12088 group (Fig. 4K). Taken together, these results suggest that DWN12088 substantially improved steatohepatitis by inhibiting NF-κB-mediated inflammatory responses.

DWN12088 reduces lipoapoptosis and hepatic injury by blocking ER stress-related signaling cascades in palmitic acid-stimulated hepatocytes and liver of MCD-diet mice

Activation of PA-mediated ER stress signal pathway is implicated in apoptosis via lipotoxicity [39]. We first investigated inhibitory effects of DWN12088 on the PA-mediated ER stress and apoptotic signaling pathways. PA-induced PERK and eIF2α phosphorylation, and ATF4 and CHOP expression were significantly inhibited by DWN12088. In addition, the reduction in the level of anti-apoptotic protein Bcl2 and increase in the levels of proapoptotic proteins, such as BCL2 associated X, apoptosis regulator (Bax) and active-caspase 3 in PA-stimulated cells, were dose-dependently reversed by DWN12088, resulting in the recovery of poly(ADP-ribose) polymerase (PARP) cleavage (Fig. 5A). Furthermore, similar results were confirmed by active-caspase 3-targeted immunofluorescence staining and TUNEL assay (Fig. 5B and C). To evaluate whether DWN12088 inhibited MCD-diet-induced hepatic injury, we first confirmed inhibitory effects of DWN12088 on the MCD-diet-mediated ER stress and proapoptotic signaling cascades in the liver tissues. As expected, compared to that with saline treatment, DWN12088 treatment markedly decreased MCD-diet-induced phosphorylation and expression of ER stress and proapoptotic signal modulators in the liver tissues (Fig. 5D). Immunofluorescence staining also showed similar results (Fig. 5E). Furthermore, MCD-diet-increased circulating ALT/AST levels and percentages of TUNEL-positive hepatocytes were drastically diminished by DWN12088 treatment (Fig. 5F and G). Moreover, H&E staining have shown that saline-treated MCD-diet group had increased the total NAS. In contrast, the NAS scores were markedly diminished in the DWN12088-treated MCD-diet group (Fig. 5H). Hence, it can be said that DWN12088 treatment prevents hepatic injury by inhibiting ER stress-mediated signaling pathway.

DISCUSSION

HF is a bioactive agent that blocks the catalytic activity of PRS, which involves the loading of proline to tRNA during the translation of proline-rich profibrotic genes, resulting in the suppression of collagen and fibronectin gene expressions [26]. DWN12088, a first-in-class PRS inhibitor, is known to be an anti-fibrotic agent that improves IPF by inhibiting the synthesis of collagen [40], but to date, no studies have been reported on its efficacy in improving hepatic injury caused by NASH. In this study, we first investigated whether DWN12088 inhibits TGFβ-induced expressions of profibrotic genes, such as collagen I, fibronectin, and α-SMA, in human HSCs LX-2. As expected, the DWN12088 treatments significantly decreased the expressions of profibrotic genes. In addition, DWN12088 treatment downregulated TGFβ-mediated activation of TGFβR1/Smad2/3 and TGFβR1/EPRS/STAT6 axis signaling. Similar results were found with mouse normal hepatocytes AML12. Consistent with our findings, a previous study demonstrated that HF and EPRS-targeting shRNA suppressed TGFβR1/Smad2/3 and TGFβR1/EPRS/STAT6 signaling pathways, leading to the downregulation of collagen 1 and fibronectin expressions in TGFβ-treated LX-2 cells and CCl4-treated mouse liver [37]. In addition, we showed that DWN12088 treatment attenuates MCD-diet-induced expressions of collagen 1, fibronectin, and α-SMA. Taken together, these finding indicate that DWN12088 plays an important role in the inhibition of hepatic fibrosis.

A recent study reported that the expressions of glucose transporter 1 (GLUT1) and hexokinase-2 were suppressed by the inhibition of AKT/mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in HF-treated colorectal cancer cells, resulting in diminished glucose uptake [41]. The mechanism by which HF inhibits lipogenesis remains unclear. In particular, several side effects, such as organ failure and gastrointestinal toxicity have been reported with the use of high-dose HF, and for this reason, it is difficult to study this agent in vivo [27]. Interestingly, we found that DWN12088 downregulates two distinct signaling cascades, ROCK/AMPK/SREBP-1c and PERK/eIF2α/ATF4/CHOP, resulting in suppressed lipid accumulation and lipoapoptosis in PA-stimulated AML12 cells in the liver of the MCD-diet mice. These findings are supported by the results of previous studies, namely that ROCK knockdown reverses the downregulation of AMPK phosphorylation, leading to inhibitions of SREBP-1c and FAS expressions in PA-stimulated L6 myotube cells [12]. Moreover, PA-activated ER stress signaling cascades, namely the PERK/eIF2α/ATF4/CHOP axis, were decreased by it targeting siRNA, resulting in inhibited anti-apoptotic Bcl2 expression [19]. Our results indicate that DWN12088 markedly inhibits hepatic lipoapoptosis by promoting lipid accumulation via downregulation of ROCK-and ER stress-mediated signaling cascades.

Since the activation of AMPK signaling suppresses NF-κB-mediated activation of inflammatory responses [38], PA may be inhibited by the phosphorylation of AMPKα on Thr172 via an increase in ROCK expression, resulting in elevated activation of NF-κB. Our data showed that DWN12088 treatment decreased PA-mediated NF-κB activation by inhibiting the downstream ROCK signaling cascades, which is consistent with the results of previous studies [12,38]. The free NF-κB dissociated from the NF-κB/IκBα inactive complex can be phosphorylated at Ser 536 and Ser 276 in the cytosol and nuclei, respectively [42]. Our results showed that PA increased but DWN12088 inhibited the phosphorylation of NF-κB p65 (Ser536, Ser276) and expressions of proinflammatory genes, such as Tnfa, Il1b, Il6, and Ccl2. Similar results were confirmed in DWN12088-treated MCD-diet fed mice. Taken together, these results suggest that DWN12088 significantly improved steatohepatitis by inhibiting NF-κB-mediated inflammatory responses.

In summary, to our knowledge, our work demonstrates for the first time that DWN12088 was safer at high doses than HF. As expected, DWN12088 suppressed hepatic fibrosis by inhibiting TGFβR1/Smad2/3 and TGFβR1/EPRS/STAT6 axis signaling. Furthermore, DWN12088 markedly attenuated hepatic lipid accumulation and lipotoxicity by inhibiting the ROCK/AMPK/SREBP-1c and PERK/eIF2α/ATF4/CHOP axis signaling pathways. We also showed that DWN12088 markedly reduced steatohepatitis by inhibiting the NF-κB-mediated inflammatory responses. In confirmation, AMPK inhibitor (compound C) treatment blocked the anti-steatosis and anti-inflammatory effects of DWN12088 (Supplementary Fig. 5).

Steatosis and inflammation are suppressed through general control nonderepressible 2 (GCN2)/mTORC1 signaling through PRS inhibitor [43]. We therefore speculate that the anti-steatosis and anti-inflammatory effects by DWN12088 are through the inhibition of PRS. The effects of DWN12088 on ROCK/AMPK/SREBP-1c and NF-κB signaling were confirmed in relation to inhibition of steatosis and inflammation respectively. Based on the above-mentioned findings, the beneficial effect of DWN12088 goes beyond its specific PRS inhibition. It also prevents fat accumulation and fat toxicity both in vivo and in vitro, thus preventing the onset of liver fibrosis. Therefore, our findings provide substantive evidence that DWN12088 plays a pivotal role in alleviating hepatic fibrosis, and we provide evidence that it shows great potential to be developed as a new integrated therapeutic agent.

SUPPLEMENTARY MATERIALS

NOTES

CONFLICTS OF INTEREST

Drug Discovery Center, Daewoong Pharmaceutical provided us with DWN12088 for this study. No potential conflict of interest relevant to this article was reported.

AUTHOR CONTRIBUTIONS

Conception or design: D.K.L., J.S.P., C.H.C.

Acquisition, analysis, or interpretation of data: D.K.L., S.H.J., E.S.L., K.B.H., N.W.P., D.H.K., S.I.P.

Drafting the work or revising: D.K.L., S.H.J.

Final approval of the manuscript: all authors.

FUNDING

This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No, 2021R1A2B5B01002354). This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No, 2022R1I1A1A01068782), this work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No, 2020R1A5A8019180).

Acknowledgements

DWN12088 used in this study was provided by Drug Discovery Center, Daewoong Pharmaceutical. We thank Dr. CH Lee for the discussions and recommendations for our research. Part of our study was also presented in oral presentation from at the 35th Spring Conference of the Korean Diabetes Association, Gyeongju, Korea, May 12 to 14, 2022.

Fig. 1.

Comparative cytotoxic effects of DWN12088 and halofuginone (HF) in mouse normal hepatocytes alpha mouse liver 12 (AML12) and human hepatic stellate cells (HSCs) LX-2. Cells were treated with various concentrations of DWN12088 (DWN) or HF. After 24 hours, (A, B) cell viability was determined by MTT assay (n=3), and (C, D) active-caspase 3 expressions were measured by immunofluorescence staining with Alexa Fluor 647-labelled active-caspase 3 and nuclei were stained with 4ʹ,6-diamidino2-phenylindole (DAPI). Scale bar: 20 μm. The number of active-caspase 3 expressing cells was counted in four fields per sample (n=3). Statistical significance was calculated using unpaired two-tailed t-test (A, B) and one-way analysis of variance (ANOVA) (C, D) followed by the Holm-Sidak post hoc test. All data are shown as the mean±standard deviation. ns, not statistically significant. ^aP<0.01, ^bP<0.001.

Fig. 2.

Effects of DWN12088 on hepatic fibrosis. LX-2 cells were stimulated with indicated concentrations of DWN12088 (DWN) for 2 hours, followed by stimulation with 2 ng/mL transforming growth factor β (TGFβ) for 24 hours. (A) Protein levels of collagen I, fibronectin, and α-smooth muscle actin (α-SMA) were determined by immunoblotting. (B) Profibrotic marker collagen I and fibronectin expressions were measured by immunofluorescence staining with Alexa Flour 488-labelled collagen I or fibronectin, and nuclei were stained with 4ʹ,6-diamidino-2-phenylindole (DAPI). Scale bar: 20 μm. (C) Relative mRNA levels of profibrotic genes were analyzed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n=3). Male C57BL/6J mice (9 weeks old) were fed a chow diet or methionine-choline deficient (MCD) diet, which was treated with or without DWN12088 (10 mg/kg, every 2 days apart, n=10 mice per group) for 6 weeks. (D) Hydroxyproline contents were measured in hepatic tissues (n=10 mice per group). (E) Masson’s trichrome staining was used to detect the collagen level of liver tissues. Distribution of blue-stained collagen fibers were analyzed in four fields per slide section (n=4). (F) Protein levels of profibrotic genes were measured by immunoblotting of liver tissues. (G) Relative mRNA levels of collagen I a1 (Col1a1), collagen I a2 (Col1a2), Col1a1 and fibronectin (Fn1), and actin alpha 2 (Acta2) were determined by qRT-PCR (n=10 mice per group). Statistical significance was calculated using one-way analysis of variance (ANOVA) (B, C) and two-way ANOVA (D, E, G) followed by the Holm-Sidak post hoc test. All data are presented as the mean±standard deviation. ns, not statistically significant; Veh, vehicle. ^aP<0.05, ^bP<0.01, ^cP<0.001.

Fig. 3.

Effects of DWN12088 on hepatic steatosis. Alpha mouse liver 12 (AML12) cells were treated with indicated concentrations of DWN12088 (DWN) for 2 hours, followed by stimulation with 250 μM palmitic acid (PA) for 24 hours. (A) 3.7% formaldehyde-fixed cells were stained with Oil red O, and (B) intracellular triglyceride (TG) concentrations were determined in cell lysates (n=3). The numbers of Oil red O-stained cells were counted in four fields per sample (n=3). Scale bar: 20 μm. (C) Phosphorylation and protein expression levels of lipogenesis-related signaling molecules were examined by immunoblotting. Mice were fed a chow diet or methionine-choline deficient (MCD) diet, which was treated with or without DWN12088 for 6 weeks. (D, E) Photographs of liver, liver weight (g), body weight (g), and liver/body weight ratio (%) are shown. (F, G) Liver sections were stained with Oil red O and labelled with boron-dipyrromethene (BODIPY) and 4ʹ,6-diamidino-2-phenylindole (DAPI) (n=5 mice per group). (H) Levels of hepatic TG, serum TG, total cholesterol, and serum free fatty acid (FFA) were measured by enzyme-linked immunosorbent assay (ELISA) (n=10 mice per group). (I, J) Phosphorylation or protein levels of Rho-kinase 1 (ROCK1) signaling cascade molecules and lipogenesis marker genes were determined by immunoblotting. Statistical significance was calculated using one-way analysis of variance (ANOVA) (A, B) and two-way ANOVA (E–I) followed by the Holm-Sidak post hoc test. All data are presented as the mean±standard deviation. Veh, vehicle; AMPK, AMP-activated protein kinase; SREBP-1c, sterol regulatory element-binding protein-1c; FAS, fatty acid synthase; ADRP, adipophilin; PPARα, peroxisome proliferator-activated receptor α; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1α; ns, not statistically significant. ^aP<0.05, ^bP<0.001.

Fig. 4.

Effects of DWN12088 on steatohepatitis. RAW264.7 cells were incubated with indicated concentrations of DWN12088 (DWN) for 2 hours prior to treatment with 250 μM of palmitic acid (PA) for 24 hours. (A) Rho-kinase 1 (ROCK1) expression and AMP-activated protein kinase (AMPK) phosphorylation were measured by immunoblotting. RAW264.7 cells were stimulated with PA alone or in combination with DWN (10 μM) in the presence or absence of MG132 (1 μM) for 24 hours to assess (B, C) phosphorylation and degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) and translocation and phosphorylation of nuclear factor-κB (NF-κB) p65. (D) NF-κB-Luc. Activity was analyzed in cell lysates (n=3). (E) Nuclear translocation of NF-κB p65 was measured by immunofluorescence staining with Alexa Fluor 647-labelled NF-κB p65. Nuclei were stained with 4ʹ,6-diamidino-2-phenylindole (DAPI). (F) mRNA expression levels of pro- and anti-inflammatory genes were quantified by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n=3). Chow- or methionine-choline deficient (MCD)-diet mice administrated with or without DWN12088 for 6 weeks. (G) Macrophages marker F4/80 expressions were determined by immunofluorescence staining with Alexa Fluor 488-labelled F4/80 and DAPI in liver sections. Relative F4/80-positive cells were quantitated in four fields per slide section (n=4). (H) EGF-like module-containing mucin-like hormone receptor-like 1 (Emr1) mRNA expression was analyzed by qRT-PCR (n=10 mice per group). (I) Degradation of IκBα and (J) phosphorylation of NF-κB p65 (serine 536, serine 276) were measured by immunoblotting of liver tissues. (K) Relative mRNA levels of inflammatory genes were quantified by qRT-PCR (n=10 mice per group). Chow- or MCD-diet mice administrated with or without DWN12088 for 6 weeks. Statistical significance was calculated using one-way analysis of variance (ANOVA) (A, D, F) and two-way ANOVA (G, H, K) followed by the Holm-Sidak post hoc test. All data are presented as the mean±standard deviation. Veh, vehicle; Tnfa, tumor necrosis factor-α; Il1b, interleukin 1β; Arg1, arginase 1; ns, not statistically significant. ^aP<0.05, ^bP<0.01, ^cP<0.001.

Fig. 5.

Effects of DWN12088 on lipoapoptosis and hepatic injury. Alpha mouse liver 12 (AML12) cells were stimulated with indicated concentrations of DWN12088 for 2 hours, followed by treatment with 250 μM of palmitic acid (PA) for 24 hours. (A) Phosphorylation and protein expression levels of endoplasmic reticulum (ER) stress- and apoptosis-related signaling molecules were examined by immunoblotting. (B) Active-caspase 3 expressions were determined by immunofluorescence staining with Alexa Fluor 647-labelled active-caspase 3 and 4ʹ,6-diamidino-2-phenylindole (DAPI). (C) Apoptotic cells were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Relative active-caspase 3- and TUNEL-positive cells were quantitated in four fields per sample (n=4). Chow- or methionine-choline deficient (MCD)-diet mice treated with or without DWN12088 for 6 weeks. (D) Phosphorylation and expression of ER stress- and apoptotic-related proteins were measured by immunoblotting. (E) Active-caspase 3-expressing hepatocytes were confirmed by immunofluorescence staining with Alexa Fluor 647-labelled active-caspase 3 and DAPI in liver sections. The numbers of active-caspase 3-positive cells were counted in four fields per tissue slide section (n=4). (F) Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured by enzyme-linked immunosorbent assay (ELISA) (n=10 mice per group). (G) Apoptotic hepatocytes were confirmed by TUNEL assay, and TUNEL-positive cells were quantitated in four fields per slide section (n=4). (H) Liver sections were stained with hematoxylin and eosin (H&E). Scale bar: 50 μm. Total nonalcoholic fatty liver disease (NAFLD) activity score (NAS) score was calculated as a sum of the scores of steatosis, lobular inflammation, and hepatocyte ballooning in H&E-stained liver tissues (n=8). Statistical significance was calculated using one-way analysis of variance (ANOVA) (B, C) and two-way ANOVA (E–H) followed by the Holm-Sidak post hoc test. All data are displayed as the mean±standard deviation. PERK, protein kinase R-like endoplasmic reticulum kinase; eIF2α, α subunit of eukaryotic initiation factor 2; ATF4, activating transcription factor 4; CHOP, C/EBP-homologous protein; Bcl2, B-cell lymphoma 2; PARP, poly(ADP-ribose) polymerase; Veh, vehicle; ns, not statistically significant. ^aP<0.05, ^bP<0.01, ^cP<0.001.

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